Role of Abscisic Acid in Strigolactone-Induced Salt Stress

Ren et al. BMC Plant Biology (2018) 18:74 https://doi.org/10.1186/s12870-018-1292-7

RESEARCH ARTICLE Open Access Role of abscisic acid in strigolactone- induced salt stress tolerance in arbuscular mycorrhizal Sesbania cannabina seedlings Cheng-Gang Ren, Cun-Cui Kong and Zhi-Hong Xie*

Abstract Background: Strigolactones (SLs) are considered to be a novel class of phytohormone involved in plant defense responses. Currently, their relationships with other plant hormones, such as abscisic acid (ABA), during responses to salinity stress are largely unknown.

Results: In this study, the relationship between SL and ABA during the induction of H2O2 – mediated tolerance to salt stress were studied in arbuscular mycorrhizal (AM) Sesbania cannabina seedlings. The SL levels increased after ABA treatments and decreased when ABA biosynthesis was inhibited in AM plants. Additionally, the expression levels of SL-biosynthesis genes in AM plants increased following treatments with exogenous ABA and H2O2. Furthermore, ABA-induced SL production was blocked by a pre-treatment with dimethylthiourea, which scavenges H2O2. In contrast, ABA production was unaffected by dimethylthiourea. Abscisic acid induced only partial and transient increases in the salt tolerance of TIS108 (a SL synthesis inhibitor) treated AM plants, whereas SL induced considerable and prolonged increases in salt tolerance after a pre-treatment with tungstate. Conclusions: These results strongly suggest that ABA is regulating the induction of salt tolerance by SL in AM S. cannabina seedlings. Keywords: Strigolactones, Abscisic acid ·arbuscular mycorrhizal, Sesbania cannabina, Photosynthesis, Salt stress

Background cannabina, which is a soil-improving legume, is highly Saline-alkali stress is a serious ecological problem that adaptable to different adverse climatic conditions, including negatively impacts plant survival, development, and prod- salinity, drought, and waterlogging stresses. Therefore, de- uctivity [1]. To survive such stress, plants have established veloping S. cannabina–AM fungi symbiotic relationships beneficial associations with several microorganisms present might represent a good strategy for improving the fertility in the rhizosphere that can alleviate the stress symptoms of saline soils to enable the growth of agriculturally import- [2]. One of the most intensively studied and widespread ant crops [6]. mutualistic plant–microorganism associations involves Establishing a functioning symbiotic relationship arbuscular mycorrhizal (AM) fungi. About 80% of terres- with an AM fungus requires a precise coordination trial plants, including most leguminous species, are able to between the partners based on a finely regulated establish this type of symbiotic relationship with fungi of molecular dialogue [7]. The molecular dialogue oc- the division Glomeromycota [3]. Indeed, AM symbiosis in- curring during the so-called pre-symbiotic stage creases resistance to soil salinity in diverse host plants, such starts when the host plant produces and exudes stri- as maize, tomato, and lettuce [4], although the underlying golactones (SLs) into the rhizosphere. Strigolactones mechanisms are not well characterized [5]. Sesbania are recognized by AM fungi with an uncharacterized receptor that stimulates hyphal growth and branch- * Correspondence: [email protected] ing, thereby increasing the probability of encounter- Key Laboratory of Biology and Utilization of Biological Resources of Coastal Zone, Yantai Institute of Coastal Zone Research, Chinese Academy of ing the host roots [8]. In accordance with their role Sciences, Yantai 264003, China as signaling molecules in the rhizosphere, SLs are

© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ren et al. BMC Plant Biology (2018) 18:74 Page 2 of 10

mainly produced in the roots and have been de- induced increase in SL production in response to salt tected in the root extracts of monocotyledonous and stress conditions. dicotyledonous plants [9]. Strigolactones are biosyn- thetically derived from carotenoids [10]viaasequen- Results tial oxidative cleavage by two carotenoid cleavage Effects of ABA levels on strigolactone accumulation dioxygenases (CCD7 and CCD8), and are apocarote- In S. cannabina seedlings inoculated with an AM fungus, noids [11], as is abscisic acid (ABA). Once synthe- the root colonization rate steadily increased as seedlings grew sized, SLs are transported acropetally to the shoot or and differed significantly between sampling times (Table 1). areexudedintotherhizospherebytheABCtrans- Additionally, the ABA concentration was the highest in salt porter PDR1, which was first identified in Petunia treated S. cannabina seedlings and it was higher in salt- hybrida [12].TheF-boxproteinMAX2andtheα/β- treated AM S. cannabina seedlings than in the water-treated fold hydrolase D14/DAD2 are the main candidate controls (Fig. 1), indicating that salt stress may have triggered components of the SL-perception complex in higher ABA biosynthesis both in AM and non-AM S. cannabina plants [13, 14]. Additionally, MAX2 has diverse seedlings. Moreover, a bioassay of SL showed that the P. roles, including in plant responses to abiotic stress ramosa germination rate increased significantly in response [15]. The importance of SLs during the initial stages to the root extract of salt-stressed AM S. cannabina seed- of mycorrhizal colonizationsiswidelyrecognized. lings [17], as did the endogenous SL levels (m/z 383, m/z356, Moreover, emerging evidence indicates that SLs may and m/z 317) (Additional file 1:TableS1). also affect the subsequent steps of the symbiotic relation- To determine whether ABA affects SL generation, we ship developing in response to environmental stimuli, examined the changes in SL levels in AM S. cannabina such as salt and drought stresses [4, 16]. We recently ob- seedlings after ABA and tungstate treatments. As shown served that SLs can induce salt stress tolerance in AM S. in Fig. 4b, ABA did not accumulate in seedlings treated cannabina seedlings that is regulated by the generation of with tungstate, which prevents the formation of ABA H2O2 as a signaling molecule [17]. from ABA aldehyde by inhibiting ABA-aldehyde oxidase Abscisic acid is an important plant hormone with a [24]. First, the ABA treatment increased SL levels in AM critical role in the regulation of salt stress responses seedlings but not in non-AM seedlings. Second, the ger- [18]. Exposure to stresses, such as salinity, induces the mination bioassay revealed that SL accumulated in salt- accumulation of ABA, resulting in increased tolerance stressed AM S. cannabina seedlings 10 days after the [19] . Several lines of evidence have revealed that ABA ABA treatment. However, this ABA-induced increase induces the accumulation of H2O2, which plays an was not observed in seedlings pre-treated with 5 μM important role in ABA signaling [20] . Moreover, SLs TIS108, a SL synthesis inhibitor (Fig. 2). These results and ABA are critical for the regulation of salt stress suggested that ABA was important for the AM fungus- responses and the establishment of symbiotic relation- induced SL synthesis under salt stress conditions. In ships between host plants and AM fungi [4, 21]. Recent contrast, when S. cannabina seedlings were treated with studies unveiled the links between ABA and SLs in a an ABA synthesis inhibitor tungstate, the SL content de- number of physiological processes. Exogenous ABA may creased significantly, but the SL level could be recovered enhance the accumulation of SLs, especially under stress by an ABA co-treatment. It also showed that H2O2 conditions [22]. Additional studies have demonstrated could affect SL accumulation in AM S. cannabina seed- that SL metabolism and its effects on ABA levels are lings (Fig. 2). Considered together, these results sug- seemingly opposite in roots and shoots under stress [23]. gested that ABA induced the accumulation of SLs in However, it is unclear whether SL and ABA are involved AM S. cannabina seedlings. in a synergistic effect during stress responses. These results suggest the possible relationship between SL and Abscisic acid affects the expression of strigolactone ABA during the induction of plant stress tolerance is biosynthesis/signaling genes in S. cannabina seedlings complex. To analyze the molecular mechanisms underlying the The mechanisms by which SLs enhance plant stress induction of SL synthesis in AM S. cannabina seedlings tolerance have so far been largely uncharacterized. Our Table 1 Colonization of Sesbania cannabina seedling roots by previous studies confirmed that H2O2 induces SL production and is critical for SL-induced salt stress toler- Funneliformis mosseae. Values are presented as the mean ± standard error of triplicate samples. Data were analyzed using ance in AM S. cannabina seedlings. In the present Duncan’s multiple range test at p < 0.05. WAS: weeks after study, we inoculated S. cannabina seedlings with AM sowing fungi and monitored the H O level, ABA content, and 2 2 Time 3 WAS 5 WAS 7 WAS SL accumulation to examine the role of ABA and the re- AMF colonization 8.6 ± 0.54c 26.3 ± 3.1b 45.8 ± 3.73a lationship between ABA and H2O2 in the AM fungus- Ren et al. BMC Plant Biology (2018) 18:74 Page 3 of 10

(Fig. 3). Furthermore, tungstate and DMTU, an H2O2 scavenger, suppressed the expression of CCD7 and CCD8 in salt-stressed AM S. cannabina roots. Additionally, seedlings pre-treated with TIS108, which blocks SL biosynthesis, exhibited up-regulated expression of CCD7 and CCD8. We also observed that salt stress, exogenous ABA, and H2O2 enhanced the expression of the SL-signaling gene, MAX2,inAMS. cannabina shoots. It is worthy to note that CCD7 and CCD8 mainly expressed in root tissue, while MAX2 in shoot tissue.

Interactions between ABA and H2O2 related to strigolactone-induced salt tolerance

An earlier study concluded that elevated H2O2 levels are involved in SL-induced salt stress tolerance [17]. Fig. 1 Abscisic acid (ABA) concentration at 1-day intervals in Sesba- nia cannabina seedlings under different salt concentrations with and Furthermore, several studies have revealed that ABA can without an inoculation with an arbuscular mycorrhizal fungus. Values induce the increased production of H2O2 (Kwak et al. are presented as the mean of three independent experiments. The [20]). Therefore, determining whether ABA helps to in- error bar represents the standard error crease H2O2 production in AM S. cannabina seedlings is warranted. To test this possibility, we pre-treated S. cannabina seedlings with tungstate, which prevented the ac- under salt stress, we examined the effects of ABA and cumulation of ABA and considerably decreased H2O2 H2O2 on the expression of CCD7, CCD8, and MAX2 levels in AM S. cannabina seedlings (Fig. 4). Additionally, homologs in S. cannabina seedlings. The expression ABA-induced SL production was inhibited by the pre- levels of two SL-biosynthesis genes, CCD7 and CCD8, treatment with DMTU (Fig. 2). These results implied that were up-regulated by NaCl, exogenous ABA, and H2O2 an increase in ABA content is required for H2O2 to induce SL production. In contrast, when we pre-treated seedlings with DMTU, we observed no significant effect on the increase in ABA content under salt stress conditions (Fig. 4b). Thus, ABA appears to function upstream of H2O2 in AM S. cannabina seedlings.

Dependency of ABA biosynthesis for strigolactone- induced salt tolerance To study the relationship between ABA- and SL-induced salt tolerances, we analyzed the effects of TIS108, a specific inhibitor of SL biosynthesis, and tungstate, an ABA biosynthesis inhibitor, on SL- and ABA-induced salt tolerances in AM S. cannabina seedlings. All plants not treated with salt had similar ΦPSII values (approximately 0.57), while those exposed to 200 mM NaCl had significantly decreased ΦPSII values at 2 and 8 days, especially in seedlings treated with TIS108 and tungstate (Fig. 5a, c). These results suggested that a defect in either ABA or SL accumulation de-

Fig. 2 Effect of hydrogen peroxide (H2O2) inhibitors and abscisic acid creased the salt stress tolerance of the seedlings. A pre- (ABA) inhibitors on strigolactone (SL) accumulation in arbuscular treatment with ABA or GR24 (synthetic SL analog) at mycorrhizal Sesbania cannabina seedlings after a 10-day treatment. 2 or 8 days prior to the exposure to salt stress signifi- Germination of Phelipanche ramosa seeds induced by S. cannabina cantly increased ΦPSII values in both control and seedling root extracts. Treatments comprised inhibitors (5 mM DMTU, 5 mM tungstate, and 2 μM TIS108) and 200 mM NaCl, which were AM plants. Moreover, GR24 restored the salt toler- applied 1 day before the inoculation with the arbuscular mycorrhizal ance of tungstate-treated plants, while ABA restored fungus. Values are presented as the mean of three independent the salt tolerance of TIS108-treated plants at 2 days experiments. The error bar represents the standard error. Data were after its application, but was ineffective at 8 days after ’ analyzed using Duncan s multiple range test. Different letters above its application (Fig. 5). Thus, SL was able to rescue the error bars indicate statistical significance at p < 0.05 the salt stress tolerance of ABA-deficient plants, while Ren et al. BMC Plant Biology (2018) 18:74 Page 4 of 10

Fig. 3 Relative expression levels of SL-biosynthesis/signaling genes in Sesbania cannabina seedlings after a 10-day treatment. a Relative expression levels of CCD7. b Relative expression levels of CCD8. c Relative expression levels of MAX2. The expression level of each gene was normalized against that of the S. cannabina tubulin gene [41] and is provided relative to the control expression level (= 1). Data are presented as the mean ± standard deviation of three biological replicates. Data were analyzed using Duncan’s multiple range test. Different letters above the error bars indicate statistical significance at p < 0.05

ABA could only partially and transiently rescue the defective stress tolerance of the tungstate-treated plants. This observation implied that SL might function downstream of ABA in plant stress responses. Furthermore, ABA-induced salt tolerance was effect- ively blocked by a pre-treatment with DMTU (Fig. 6), while SL-induced salt tolerance was regulated by DMTU [17]. These results suggested that H2O2 was also respon- sible for the observed ABA- and SL-induced salt tolerances.

Discussion Plants are sessile in nature. Therefore, to counter biotic and abiotic stresses, such as pathogens, drought, waterlogging, extreme temperatures, salinity, and many others, plants have evolved intricate signaling mechanisms composed of multiple components including plant hormones. Research conducted in the last decade have added SLs to the growing list of plant hormones involved in responses to environmental stresses [25]. In the present study, we revealed that SLs can be regulated by ABA production in S. cannabina seedlings (Figs 1, 2). Given the common metabolic precursor shared by ABA and SL, a potential crosstalk between these two hormones was recently proposed, although these hormones appear to be negatively correlated. For example, SL levels reportedly decreased, while ABA content increased in tomato and lettuce plants grown under drought conditions in the absence of AM symbiosis [16]. We also observed that ABA levels increased rapidly under salt stress conditions in plants not colonized by an AM fungus, whereas SL production was very low (Fig. 1). Therefore, this negative correlation might represent a general plant strategy to cope with water-related stresses in the absence of mycorrhization. Conversely, a positive correlation between ABA and SLs has been observed in stress-treated mycorrhizal plants [4, 16]. Similarly, increasing ABA and SL levels were detected in S. cannabina seedlings under salt stress conditions in the presence of the AM fungus (Fig. 1). Additionally, ABA enhanced P. ramosa germination, whereas tungstate had the opposite effect (Fig. 2). The different relationships between SL and ABA in AM and non-AM S. cannabina respectively may indicate that the ABA catabolism was Ren et al. BMC Plant Biology (2018) 18:74 Page 5 of 10

pre-treatment with DMTU (Fig. 4b). Previous investigations also concluded that ABA causes H2O2 to accumulate in the apoplast, which is dependent on NADPH oxidase and is important for ABA signaling [20, 26]. These results suggest that ABA may act upstream of H2O2 in the signaling pathway of AM S. cannabina seedlings. Furthermore, we provided evidence that H2O2 is important for ABA-induced SL production. We also observed that SL production wasconsiderablydecreasedbyapre-treatmentwith tungstate or DMTU (Fig. 2). An ABA treatment partly restored the P. ramosa germination rate in DMTU-treated mycorrhizal plantlets. Simultaneously, an H2O2 treatment significantly restored the P. ramosa germination rate of tungstate-treated mycorrhizal plantlets (Fig. 2). Taken together, these results implied that ABA induced SL accumulation in AM S. cannabina seedlings, which may be partially dependent on increased H2O2 levels. In this study, we confirmed that ABA is involved in the SL-induced salt tolerance of S. cannabina seedlings inoculated with an AM fungus. Exposure to tungstate decreased SL levels and ΦPSII values in salt-stressed AM S. cannabina seedlings (Figs 2, 5a, c). Furthermore, an ABA treatment significantly restored the P. ramosa germination rate in TIS108-treated mycorrhizal plantlets, indicating that the ABA-induced salt stress tolerance of AM S. cannabina seedlings required the accumulation of SL (Fig. 2). The relationship between SL and ABA during plant responses to abiotic stresses has been rarely studied. Many investigations have considered ABA as the ‘abiotic stress hormone’ because its biosyn- Fig. 4 Hydrogen peroxide (H2O2) concentration and abscisic acid thesis is rapidly induced by exposures to environmental (ABA) accumulation in Sesbania cannabina seedlings after 10-day stresses, especially water-related stresses such as drought treatments. a H2O2 content. b Abscisic acid level. Treatments comprised inhibitors (5 mM DMTU and 5 mM tungstate) and and salinity [27–29]. The reported increase in the ABA μ exogenous signaling molecules (10 mM H2O2 and 100 M ABA). content of AM plants subjected to abiotic stress likely Values are presented as the mean of three independent experiments. contributes to the enhanced tolerance of these plants to The error bar represents the standard error. Data were analyzed using environmental stresses [4, 30]. Additionally, increased Duncan’s multiple range test. Different letters above the error bars indicate statistical significance at p <0.05 SL levels were detected in lettuce plants grown in the presence of the AM fungus Rhizophagus irregularis under salt stress conditions [4]. A similar trend has been observed in drought-treated lettuce and tomato plants intensively altered by AM inoculation. Overall, it seem [16]. These observations combined with our results indi- that the increase in ABA concentration induced by the cate that SL-induced salt stress tolerance of AM plants AM fungus contributed to the increased accumulation of may be mediated by a complex set of signal transduction SLs in salt-stressed S. cannabina seedlings. pathways with ABA as a common signaling molecule. Our previous results suggested that H2O2-induced SL production is due to induced/activated NADPH oxidase Conclusions and is important for SL-induced salt stress tolerance. In In summary, we uncovered a dynamic interplay between this study, we confirmed that ABA has a key role in the SLs, ABA, and H2O2 produced in response to an AM production of H2O2, which regulates the accumulation of fungus that contributed to the salt stress tolerance of S. SLs. We revealed that H2O2 production was substantially cannabina seedlings. Following the perception of the ABA decreased by a pre-treatment with tungstate (Fig. 4a). signal, H2O2 was rapidly produced, which then increased Moreover, ABA accumulation was unaffected by a the accumulation of SLs, and ultimately enhanced the salt Ren et al. BMC Plant Biology (2018) 18:74 Page 6 of 10

Fig. 5 Effects of strigolactone (SL) and abscisic acid (ABA) levels on photosynthetic parameters in salt-stressed Sesbania cannabina seedlings. a Photosystem II efficiency (ΦPSII) values of S. cannabina seedlings after 2 days under salt stress conditions. b Non-photochemical quenching of chlorophyll fluorescence (NPQ) of S. cannabina seedlings after 2 days under salt stress conditions. c Photosystem II efficiency values of S. cannabina seedlings after 8 days under salt stress conditions. d Non-photochemical quenching of chlorophyll fluorescence of S. cannabina seedlings after 8 days under salt stress conditions. Exogenous signaling molecules (1 μM GR24 and 100 μM ABA) were applied 1 day before the S. cannabina seedlings were exposed to salt stress conditions. Inhibitors (5 mM tungstate and 2 μM TIS108) were applied 1 day before the exogenous signaling molecules were applied. Values are presented as the mean of six independent experiments. The error bar represents the standard error. Data were analyzed using Duncan’s multiple range test. Different letters above the error bars indicate statistical significance at p <0.05

stress tolerance of the seedlings. Additional studies are sterilized in 5% sodium hypochlorite for 5 min and needed to provide the genetic evidence of the involvement rinsed several times with distilled water. The seeds were of ABA in H2O2-induced SL generation and to identify germinated at 28 °C in distilled water and then sown in the critical signaling components between SL production trays containing autoclaved zonolite. After 2 weeks, indi- and salt stress responses in S. cannabina seedlings inocu- vidual seedlings were transferred to 1-L pots containing lated with an AM fungus. Such studies will help to eluci- autoclaved zonolite inoculated with 10 g inoculum date the molecular mechanism underlying the ABA- and (approximately 121 spores). The original inoculum [AM SL-induced salt tolerance of AM plants. fungus Funneliformis mosseae] was propagated in pot- cultured Trifolium repens plants for 8 weeks, and included Methods infected roots, hyphae, spores, and substrates. The growth Plant materials and treatments conditions were as follows: 12 h photoperiod, 25/17 °C − − Sesbania cannabina (Retz.) Pers. seeds were obtained (day/night), and light intensity of 600 μmol m 2 s 1. from the Shandong Academy of Agricultural Sciences, Three-week-old seedlings were used for all treatments Shandong, China. Before sowing, the seeds were surface- with 200 mM NaCl solutions. The reagents used as Ren et al. BMC Plant Biology (2018) 18:74 Page 7 of 10

Photosynthetic parameters Gas exchange and modulated chlorophyll fluorescence parameters were simultaneously detected using a LI- 6400XTR Portable Photosynthesis System (Li-Cor, Lincoln, NE, USA) equipped with a 6400–40 Leaf Chamber Fluorometer (Li-Cor). The leaves were incubated in darkness for 20 min before being analyzed. The minimal fluorescence level of the dark-adapted leaves was measured using a − − modulated pulse (< 0.05 μmol m 2 s 1 for 1.8 s), while the maximal fluorescence level was measured after applying a saturating actinic light pulse of − − 8000 μmol m 2 s 1 for 0.7 s. The actinic light − − intensity was increased to 1000 μmol m 2 s 1 and then maintained for about 30 min. The steady-state fluorescence yield was also recorded. A saturating actinic light pulse of − − 8000 μmol m 2 s 1 for 0.7 s was then applied to induce the maximum fluorescence yield by temporarily inhibiting photosystem II (PSII) photochemical activities. The minimum steady-state fluorescence yield was determined during a brief interruption of the actinic light irradiation in thepresenceoffar-redlight(λ = 740 nm). Finally, PSII efficiency (ΦPSII) and non-photochemical quenching of chlorophyll fluorescence were calculated according to the method described by Maxwell and Johnson [35].

Measurement of arbuscular mycorrhizal fungal colonization The percentage of roots colonized by mycorrhizal fungi was calculated using the gridline intersection method

Fig. 6 Effect of abscisic acid (ABA) and H2O2 inhibitors on the [36] after samples were stained with trypan blue [37]. photosynthetic parameters of arbuscular mycorrhizal Sesbania

cannabina seedlings under salt stress conditions. a Photosystem II Measurement of H2O2 Φ efficiency ( PSII) values of S. cannabina seedlings after 8 days under Seedlings were harvested 10 days after treatments to salt stress conditions. b Non-photochemical quenching of chlorophyll fluorescence (NPQ) of S. cannabina seedlings after 8 days under salt measure the H2O2 content. The H2O2 concentration stress conditions. Exogenous signaling molecules and inhibitors was determined by monitoring the absorbance of titanium comprised 100 μM ABA and 5 mM DMTU. Values are presented as the peroxide at 415 nm following the method of Brennan and mean of six independent experiments. The error bar represents the Frenkel [38]. One unit of H2O2 was defined as the chemi- ’ standard error. Data were analyzed using Duncan s multiple range test. luminescence produced by the internal standard of 1 μM Different letters above the error bars indicate statistical significance − 1 at p < 0.05 H2O2 g fresh weight.

Extraction of ABA and HPLC analysis specific scavengers or inhibitors [5 mM DMTU (H2O2 Seedlings collected for the ABA analysis were immedi- scavenger) [31], 5 mM sodium tungstate [32], and 2 μM ately frozen with liquid N2 and stored at − 80 °C. Root TIS108 (the most potent and specific SL biosynthesis in- tissue (0.5 g) was ground in a mortar and homogenized hibitor) [33]] were purchased from Sigma-Aldrich (St in 5 ml pre-cooled 80% aqueous acetone (4:1, v/v) Louis, MO, USA). Additionally, 10 mM H2O2,100μM supplemented with 10 mg/l butylated hydroxytoluene. ABA, and 1 μM GR24 (synthetic SL analog) were used as Extracts were eluted through a Sep Pak C18-cartridge exogenous signaling molecules [34]. Plantlet leaves were (PerkinElmer, Waltham, MA, USA) to remove polar sprayed with 100 μL exogenous signaling molecule or in- compounds and then purified using ethyl acetate and hibitor solution. Treatments were completely randomized NaHCO3 as described by Sweetser and Vatvars [39]. After and replicated three times. An equal volume of distilled centrifuging samples at 14,000×g for 30 min at 4 °C, the water was applied as the control treatment. Unless stated supernatant was acidified to pH 3.0 with H2SO4 and then otherwise, inhibitors were applied 24 h before the exogen- treated three times with an equal volume of ethyl ous signaling molecules were applied. acetate. The extract phase was evaporated at 40 °C Ren et al. BMC Plant Biology (2018) 18:74 Page 8 of 10

Table 2 Sequences of qRT-PCR primers used to analyze strigolactone-related genes Gene GenBank accession no. Primers ScCCD7 MF683952 5’-TGCTCCATCCACAACAGA-3′ 5’-CTCCATAAGGCCAACACC-3’ ScCCD8 MF683953 5’-TGGTCTAGCCACAAAGAA-3′ 5’-ATGAACATGGGAAAGGAT-3’ ScMAX2 MF683954 5′-TTGGATTAGGGTTGGTGA-3′ 5’-CTGAAGGTGCCGTGAGTA-3’ The nucleotide sequence reported in this table has been submitted to GenBank with accession numbers MF683952, MF683953 and MF683954

using the RE-100 rotary evaporator (Bibby Sterlin Ltd. were sealed, enclosed in polyethylene bags, and , Stone Staffordshire, UK). The dried samples were incubated at 25 °C in darkness for 7 days. The re-dissolved in chromatography-grade acetonitrile with germinated and non-germinated seeds were counted 0.1 M acetic acid and then analyzed by HPLC using a using a stereoscope. Seeds were considered germinated Hypersil-ODS C18 column (4.6 mm × 250 mm, 5 μm). if the radicle protruded through the seed coat. The mobile phase comprised acetonitrile: 0.03 M acetic acid (30:70, v/v) at a flow rate of 1 ml/min. Abscisic acid Quantitative real-time PCR was detected spectrophotometrically at 254 nm at 25 °C. Root samples from three seedlings were pooled to represent The retention time was 13.37 min. Abscisic acid standard a single biological replicate. Total RNA was extracted from curves (20–3000 ng/ml) were used to quantify ABA con- the pooled root samples using TRIzol (Invitrogen, Carlsbad, tents (R2 =0.991). CA, USA) following the manufacturer’s instructions. Three biological replicates were prepared. The extracted RNA was Extraction of strigolactones and quantification by a used as the template for reverse transcription reactions germination bioassay completed with the GoScript™ Reverse Transcription Root tissue (0.5 g) was collected and ground in a mortar System (Promega, Madison, WI, USA). Gene-specific with liquid nitrogen and then treated with 1 ml ethyl primers were designed using the Primer Premier program acetate in a 3-ml glass tube. The tubes were vortexed (version 5.0) (Premier Biosoft International). The quantita- and sonicated for 10 min in a Branson 3510 ultrasonic tive real-time (qRT)-PCR analysis was conducted with an bath (Branson Ultrasonics, Danbury, CT, USA). Samples iCycler iQ Real-time PCR Detection System (Bio-Rad) with were then centrifuged at 4000×g for 5 min in an MSE three technical replicates. Each reaction was completed in a Mistral 2000 centrifuge (Mistral Instruments, Leicester, total volume of 20 μl, which included 0.2 μM primer pairs, UK). The organic phase was carefully transferred to 1- 2 μldilutedcDNA,and10μl 2× SYBR Green PCR Master ml glass vials and stored at − 20 °C until used in the ger- Mix (TaKaRa Bio Inc., Dalian, China). The PCR program mination bioassays. was as follows: 95 °C for 30 s; 40 cycles of 95 °C for 5 s, 58 ° Strigolactones were identified by liquid chromatography– C for 15 s, and 72 °C for 20 s. Tubulin genes were used as tandem mass spectrometry using a Quattro LC mass spec- the internal controls and relative gene expression levels −ΔΔ trometer (Micromass, Manchester, UK) equipped with an were calculated using the 2 Ct method [41]. The electrospray source as previously described (Kong et al. [17]). sequences of the qRT-PCR primers are listed in Table 2. The germination bioassays with Phelipanche ramosa seeds were conducted according to the method devel- Statistical analysis oped by Yoneyama [40]. Approximately 20 surface-steril- All data were analyzed using Microsoft Excel (Redmond, ized P. ramosa seeds were placed on 6-mm glass fiber WA, USA) and the values for each treatment are presented discs (Whatman) and about 90 discs were incubated in a as the mean ± standard deviation of three replicates. A Petri dish (9 cm diameter) lined with filter paper moist- one-way analysis of variance was completed with the SPSS ened with 6 ml sterile Milli-Q water. The seeds required Statistics 17.0 software (SPSS, Inc., Chicago, IL, USA). a 12-day pre-conditioning incubation at 21 °C in dark- Duncan’s multiple range test was used to compare mean ness before they became responsive to the germination values at the p < 0.05 significance level. stimulants. A sterile Petri dish (5 cm diameter) was lined with filter paper moistened with 50 μl S. cannabina root extract, which was allowed to evaporate before the Additional file pre-conditioned seeds were added. The seeds were μ Additional file 1: Table S1 Effect of different treatments on the relative then treated with 650 l sterile Milli-Q water. The abundances of three SLs candidates m/z 383, m/z 356 and m/z 317 in S. − 6 synthetic germination stimulant GR24 (10 M) and cannabina seedlings extract.Data were analyzed using Duncan’s multiple demineralized water were included as positive and range test. Different letters indicate statistical significance at p < 0.05. (XLSX 13 kb) negative controls in each bioassay. The Petri dishes Ren et al. BMC Plant Biology (2018) 18:74 Page 9 of 10

Abbreviations 10. López-Ráez JA, Charnikhova T, Gómez-Roldán V, Matusova R, Kohlen W, De ABA: Abscisic acid; AM: Arbuscular mycorrhizal; CCD: Carotenoid cleavage Vos R, et al. Tomato strigolactones are derived from carotenoids and their dioxygenases; DMTU: Dimethylthiourea; m/z: Mass to charge ratio; biosynthesis is promoted by phosphate starvation. New. Phytologia. MAX2: More axillary growth2; NPQ: Non-photochemical quenching of 2008;178:863–74. chlorophyll fluorescence; SL: Strigolactone; TIS108: 6-phenoxy-1-phenyl-2-(1H- 11. Walter MH, Strack D. Carotenoids and their cleavage products:biosynthesis 1,2,4-triazol-1-yl) hexan-1-one; ΦPSII: Photosystem II efficiency and functions. Nat Prod Rep. 2011;28:663–92. 12. Kretzschmar T, Kohlen W, Sasse J, Borghi L, Schlegel M, Bachelier JB, et al. A Acknowledgements petunia ABC protein controls strigolactone-dependent symbiotic signalling We thank Professor Yong-Qing Ma (Northwest Agriculture and Forestry and branching. Nature. 2012;483:341–4. University) for supplying the Phelipanche ramosa seeds. 13. Stirnberg P, Furner IJ, Leyser HMO. MAX2 participates in an SCF complex which acts locally at the node to suppress shoot branching. Plant J. Funding 2007;50:80–94. This work was financed by the National Natural Science Foundation of China 14. Zhao LH, Zhou XE, Wu ZS, Yi W, Xu Y, Li S, et al. Crystal structures of two (31601238 and 31570063), the High-tech Industrialization Cooperation Funds phytohormone signal-transducing alpha/beta hydrolases: karrikin-signaling of Jilin province and the Chinese Academy of Science (2017SYHZ0007), KAI2 and strigolactone-signaling DWARF14. Cell Res. 2013;23:436–9. Shandong Key Research and Development Program (2016CYJS05A01–1, 15. Bu QY, Lv TX, Shen H, Luong P, Wang J, Wang Z, et al. Regulation of 2017GSF17129), Shandong Key Scientific and Technological Innovation drought tolerance by the F-box protein MAX2 in Arabidopsis. Plant Physiol. Program (2017CXGC0303), Mechanisms of marginal land productivity 2014;164:424–39. expansion and technologies of storing grain in land (KFZD-SW-112), Yantai 16. Ruiz-Lozano JM, Aroca R, Zamarreño ÁM, Molina S, Andreo-Jiménez B, Key Project of Research and Development Plan (2016ZH074). Porcel R, et al. Arbuscular mycorrhizal symbiosis induces strigolactone biosynthesis under drought and improves drought tolerance in lettuce and Availability of data and materials tomato. Plant Cell Environ. 2016;39:441–52. All data generated or analysed during this study are included in this 17. Kong CC, Ren CG, Li RZ, Xie ZH, Wang JP. Hydrogen peroxide and published article [and its Additional file]. strigolactones signaling are involved in alleviation of salt stress induced by arbuscular mycorrhizal fungus in sesbania cannabina seedlings. J Plant Authors’ contributions Growth Regul. 2017; https://doi.org/10.1007/s00344-017-9675-9 RCG designed and performed the experiment, analysed the data and wrote 18. Ha CV, Leyva-González MA, Osakabe Y, Tran UT, Nishiyama R, Watanabe Y, the manuscript. KCC perform extraction and identification the et al. Positive regulatory role of strigolactone in plant responses to drought – phytohormone using GC-MS. RCG and KCC participated in revisions of the and salt stress. PNAS. 2014;111(14):851 6. manuscript. All authors have read the manuscript, given comments and ap- 19. Zhu JK. Salt and drought stress signal transduction in plants. Annu Rev proved the final version of the manuscript. Plant Biol. 2002;53:247–73. 20. Kwak JM, Mori IC, Pei ZM, Leonhardt N, Torres MA, Dangl JL. NADPH Ethics approval and consent to participate oxidase AtrbohD and AtrbohF genes function in ROS-dependent ABA – Not applicable signaling in Arabidopsis. EMBO J. 2003;22:2623 33. 21. 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